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Studies of biosynthesis of insulin by pH-5 enzymes-microsome system from fetal dog pancreas
180
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY NOTES PN 91043
Studies on biosynthesis of insulin by pH.5 enzymes-microsome system from fetal dog pancreas Previous studies from this laboratory 1 have shown that slices from rat pancreas incorporated [z4C~amino acids into insulin. These studies were extended to study the incorporation of Ez4C]amino acids into insulin by pH-5 enzymes-microsome system prepared from fetal dog pancreas. Fetal dog pancreas in early stages of fetal development was chosen to minimize the effects of p r o t e o l y t i c enzymes 2. Pancreases were removed from pups, obtained by caesarean section of pregnant dogs in mid stage of gestation. One to two g of pancreas tissue were homogenized in phosphate buffer and pH-5 enzymes and microsomes were prepared b y procedure of HOAGLAND et al. ~ as reported previously from this laboratory for rat liver preparations 4. The pH-5 enzymes and microsomes were then incubated with [14C~amino acids adenosine triphosphate, guanosine triphosphate, phosphoenol pyruvate and pyruvate kinase (EC 2.7.I.4O) for I h at 37 ° and at the end of incubation the mixtures were extracted with acid alcohol by the procedure of Pettinga ~. Aliquots of acid-alcohol extracts were assayed for insulin like activity by the rat epididymal fat pad method as reported earlierZ, 6. Another set of aliquots was incubated in glycine buffer (pH 9.2) with and without normal serum and anti-insulin selum. The insulin antibody complex was then precipitated with sodium sulfiteL~ and assayed for radioactivity. The results of these studies are given in Table I. It can be seen in Table I that pH-5 enzymes-microsome system from fetal dog pancreas show a two-fold increase in inTABLE I INSULIN-LIKE ACTIVITY IN p H - 5 ENZYMES AND MICROSOMES PREPARED FROM FETAL DOG PANCREAS p H - 5 e n z y m e s a n d / o r microsomes were i n c u b a t e d in a t o t a l volume of 2 ml of p h o s p h a t e buffer (pH 7.6) w i t h [14C]amino acids (0.25 # m o l e each of the a m i n o acids as p r e s e n t in the insulin molecule) containing 2. ioe-2.5 • lO 6 c o u n t s / m i n , 5/zmoles adenosine t r i p h o s p h a t e , i.o/*mole guanosine t r i p h o s p h a t e , 1.25/zmoles p h o s p h o e n o l p y r u v a t e and o.I mg p y r u v a t e kinase. All results are expressed per each of the i n c u b a t i o n m i x t u r e a n d each figure is an average of four values.
System
Amount (rag)
Condition
[I-14C]glucose Units oxidized to 14C02 insulin-like (counts/rain~ activity g /at pad*)
i. 2. 3. 4. 5.
I0 io 3° 3° lO+3O
unincubated incubated unincubated incubated unincubated
72o~ 62 738~ 88 12 8 0 0 ± 2 3 0 0 15 6oo-- 185o
---o.3IiO.O2 o . 3 4 ~ 0.o3
13 ooo i 18oo
o.36:~- o.o 3
lO+3O
incubated 28 8 8 o ~ 36oo
o.78~o.o6
p H - 5 enzymes pH- 5 enzymes Microsomes Microsomes pH-5 e n z y m e s + microsomes 6. p H - 5 e n z y m e s + microsomes
14C activity bound by antiinsulin serum (counts/rain)
2oo~ 18 i26o:~i2o
8800±83 °
* Approx. 500 mg of epididymal fat p a d tissue was i n c u b a t e d for 90 nlin in 6 ml of R i n g e r b i c a r b o n a t e m e d i u m w i t h [i-l*C]glucose and 1~CO2 w a s isolated and c o u n t e d as BaCO:,.
Biochim. Biophys. Acta, 95 (1965) 18o-181
181
PRELIMINARY NOTES
sulin-like activity upon incubation with amino acids compared to unincubated preparations. These studies would indicate that a small amount of insulin synthesis was obtained in the cell free system. This is further confirmed b y the observation that large amounts of radioactivity incorporated into acid-alcohol fraction are precipitated b y anti-insulin serum. However the possibility cannot be ruled out that the observed insulin-like activity m a y be due to combination of A and B chains of insulin already present in the microsomes. Experiments are in progress to study if this is a true synthesis of insulin b y further purification of the insulin fraction. Furthermore, it was obseived that pH-5 enzymes and microsomes from pancreas tissue were easily inactivated as compared to liver preparations and careful handling of this tissue and fractions was essential to obtain the active preparations. This investigation was supported by Public Health Service Research grant No. AM-o8242 from Institute of Arthritis and Metabolism Diseases.
Department o/ Pharmacology, Indiana University Medical School, Indianapolis, Ind. (U.S.A.)
S. R. WAGLE
S. R. ~VAGLE, Biochem. Biophys. Res. Commun., 13 (1963) 175. F. G. BANTING AND C. H. BEST, J. Lab. Clin. Med., 7 (1922) 464 • M. B. HOAGLAND, E. ]3. KELLER AND P. C. ZAMECNIK, J. Biol. Chem., 218 (1956) 345. S. R. ~VAGLE, Arch. Biochem. Biophys., lO2 (1963) 373. C. W. PETTINGA, Biochem. Prepn., 6 (196o) 28. A. E. RENOLD, D. B. MARTIN, Y. M. DAGENAIS, J. STEINKE, 1~. J. NICKERSON AND M. C. SHEPS, J. Clin. Invest., 39 (196o) 1487. 7 G. M. ORODSKY AND E. H. FORSHAM, J. Clin. Invest., 39 (196o) lO7 o.
I 2 3 4 .5 6
Received October 2Ist, 1964 Biochim. Biophys. Acta, 95 (1965) 18o-181
PN
91044
Hydroxyureo: A specific inhibitor of deoxyribonucleic acid synthesis This laboratory has been interested in studying the mode of action of various metabolic inhibitors z-5. Our interest in hydroxylamine 3 led us to investigate the properties of the anti-leukemic agent hydroxyurea, especially because hydroxylamine has been claimed to be a degradation product of hydroxyurea 6. The current study is concerned with the effects of hydroxyurea on Escherichia coli C6oo, a substrain of E. coli KI2. The effectiveness of hydroxyurea on various macromolecular syntheses and processes was measured by determining the concentration of hydroxyurea required to limit increases of these functions b y 50 % when compared to untreated control cultures. The data illustrated in Fig. I indicate that hydroxyurea has a bacteriostatic rather than a bactericidal effect. A concentration of 0.032 M hydroxyurea was sufficient to cause 50 % reduction in the increase in number of viable bacteria. On the other hand, the increase in turbidity of the culture, which is an index of protein Biochim. Biophys. Acta, 96 (1965) I 8 I - I 8 3
BIOCHIMICA ET BIOPHYSICA ACTA
PRELIMINARY NOTES PN 91043
Studies on biosynthesis of insulin by pH.5 enzymes-microsome system from fetal dog pancreas Previous studies from this laboratory 1 have shown that slices from rat pancreas incorporated [z4C~amino acids into insulin. These studies were extended to study the incorporation of Ez4C]amino acids into insulin by pH-5 enzymes-microsome system prepared from fetal dog pancreas. Fetal dog pancreas in early stages of fetal development was chosen to minimize the effects of p r o t e o l y t i c enzymes 2. Pancreases were removed from pups, obtained by caesarean section of pregnant dogs in mid stage of gestation. One to two g of pancreas tissue were homogenized in phosphate buffer and pH-5 enzymes and microsomes were prepared b y procedure of HOAGLAND et al. ~ as reported previously from this laboratory for rat liver preparations 4. The pH-5 enzymes and microsomes were then incubated with [14C~amino acids adenosine triphosphate, guanosine triphosphate, phosphoenol pyruvate and pyruvate kinase (EC 2.7.I.4O) for I h at 37 ° and at the end of incubation the mixtures were extracted with acid alcohol by the procedure of Pettinga ~. Aliquots of acid-alcohol extracts were assayed for insulin like activity by the rat epididymal fat pad method as reported earlierZ, 6. Another set of aliquots was incubated in glycine buffer (pH 9.2) with and without normal serum and anti-insulin selum. The insulin antibody complex was then precipitated with sodium sulfiteL~ and assayed for radioactivity. The results of these studies are given in Table I. It can be seen in Table I that pH-5 enzymes-microsome system from fetal dog pancreas show a two-fold increase in inTABLE I INSULIN-LIKE ACTIVITY IN p H - 5 ENZYMES AND MICROSOMES PREPARED FROM FETAL DOG PANCREAS p H - 5 e n z y m e s a n d / o r microsomes were i n c u b a t e d in a t o t a l volume of 2 ml of p h o s p h a t e buffer (pH 7.6) w i t h [14C]amino acids (0.25 # m o l e each of the a m i n o acids as p r e s e n t in the insulin molecule) containing 2. ioe-2.5 • lO 6 c o u n t s / m i n , 5/zmoles adenosine t r i p h o s p h a t e , i.o/*mole guanosine t r i p h o s p h a t e , 1.25/zmoles p h o s p h o e n o l p y r u v a t e and o.I mg p y r u v a t e kinase. All results are expressed per each of the i n c u b a t i o n m i x t u r e a n d each figure is an average of four values.
System
Amount (rag)
Condition
[I-14C]glucose Units oxidized to 14C02 insulin-like (counts/rain~ activity g /at pad*)
i. 2. 3. 4. 5.
I0 io 3° 3° lO+3O
unincubated incubated unincubated incubated unincubated
72o~ 62 738~ 88 12 8 0 0 ± 2 3 0 0 15 6oo-- 185o
---o.3IiO.O2 o . 3 4 ~ 0.o3
13 ooo i 18oo
o.36:~- o.o 3
lO+3O
incubated 28 8 8 o ~ 36oo
o.78~o.o6
p H - 5 enzymes pH- 5 enzymes Microsomes Microsomes pH-5 e n z y m e s + microsomes 6. p H - 5 e n z y m e s + microsomes
14C activity bound by antiinsulin serum (counts/rain)
2oo~ 18 i26o:~i2o
8800±83 °
* Approx. 500 mg of epididymal fat p a d tissue was i n c u b a t e d for 90 nlin in 6 ml of R i n g e r b i c a r b o n a t e m e d i u m w i t h [i-l*C]glucose and 1~CO2 w a s isolated and c o u n t e d as BaCO:,.
Biochim. Biophys. Acta, 95 (1965) 18o-181
181
PRELIMINARY NOTES
sulin-like activity upon incubation with amino acids compared to unincubated preparations. These studies would indicate that a small amount of insulin synthesis was obtained in the cell free system. This is further confirmed b y the observation that large amounts of radioactivity incorporated into acid-alcohol fraction are precipitated b y anti-insulin serum. However the possibility cannot be ruled out that the observed insulin-like activity m a y be due to combination of A and B chains of insulin already present in the microsomes. Experiments are in progress to study if this is a true synthesis of insulin b y further purification of the insulin fraction. Furthermore, it was obseived that pH-5 enzymes and microsomes from pancreas tissue were easily inactivated as compared to liver preparations and careful handling of this tissue and fractions was essential to obtain the active preparations. This investigation was supported by Public Health Service Research grant No. AM-o8242 from Institute of Arthritis and Metabolism Diseases.
Department o/ Pharmacology, Indiana University Medical School, Indianapolis, Ind. (U.S.A.)
S. R. WAGLE
S. R. ~VAGLE, Biochem. Biophys. Res. Commun., 13 (1963) 175. F. G. BANTING AND C. H. BEST, J. Lab. Clin. Med., 7 (1922) 464 • M. B. HOAGLAND, E. ]3. KELLER AND P. C. ZAMECNIK, J. Biol. Chem., 218 (1956) 345. S. R. ~VAGLE, Arch. Biochem. Biophys., lO2 (1963) 373. C. W. PETTINGA, Biochem. Prepn., 6 (196o) 28. A. E. RENOLD, D. B. MARTIN, Y. M. DAGENAIS, J. STEINKE, 1~. J. NICKERSON AND M. C. SHEPS, J. Clin. Invest., 39 (196o) 1487. 7 G. M. ORODSKY AND E. H. FORSHAM, J. Clin. Invest., 39 (196o) lO7 o.
I 2 3 4 .5 6
Received October 2Ist, 1964 Biochim. Biophys. Acta, 95 (1965) 18o-181
PN
91044
Hydroxyureo: A specific inhibitor of deoxyribonucleic acid synthesis This laboratory has been interested in studying the mode of action of various metabolic inhibitors z-5. Our interest in hydroxylamine 3 led us to investigate the properties of the anti-leukemic agent hydroxyurea, especially because hydroxylamine has been claimed to be a degradation product of hydroxyurea 6. The current study is concerned with the effects of hydroxyurea on Escherichia coli C6oo, a substrain of E. coli KI2. The effectiveness of hydroxyurea on various macromolecular syntheses and processes was measured by determining the concentration of hydroxyurea required to limit increases of these functions b y 50 % when compared to untreated control cultures. The data illustrated in Fig. I indicate that hydroxyurea has a bacteriostatic rather than a bactericidal effect. A concentration of 0.032 M hydroxyurea was sufficient to cause 50 % reduction in the increase in number of viable bacteria. On the other hand, the increase in turbidity of the culture, which is an index of protein Biochim. Biophys. Acta, 96 (1965) I 8 I - I 8 3